Targeted inhibition of the ERK-MAPK pathway, upregulated in the majority of human cancers, has been hindered in the clinic by drug resistance and on-target toxicity. The MRAS-SHOC2-PP1 complex plays a key, but underexplored role in RAF-ERK pathway activation, by dephosphorylating a critical inhibitory site on RAF-kinases. In this body of work we present a preferential requirement for the SHOC2-phosphatase complex, specifically for Receptor Tyrosine Kinase (RTK), and anchorage-independent (tumorigenic) growth stimulated ERK-activation. We highlight that this context-dependent signalling bias has functional consequences in RAS-mutant cells, by specifically inhibiting anchorage-independent, but not 2D-adhered cell growth. Strikingly we show in vivo that SHOC2 deletion suppresses tumour initiation in KRAS-driven lung cancer models, and significantly extends overall survival. Additionally, SHOC2 inhibition selectively sensitizes KRAS- and EGFR-mutant Non-small cell lung carcinoma (NSCLC) cells to MEK inhibitors. Mechanistically we show this is because SHOC2 is required for feedback-induced RAF dimerization, and as such combined MEK inhibition and SHOC2 suppression leads to more potent and sustained ERK-pathway repression, driving a BIM-dependent apoptosis. Crucially, systemic SHOC2 ablation in adult mice is relatively well tolerated compared with other, core, ERK-pathway signalling nodes. These results present a rationale for the generation of SHOC2 targeted therapies, both as a monotherapy, and to widen the therapeutic index of MEK inhibitors.

Genetic variants that affect megakaryopoiesis and platelet formation result in inherited bleeding and platelet disorders (BPD). Only 40-60% of cases will receive a diagnosis indicating the pathway at fault [1, 2]. This thesis is the result of work undertaken to discover novel variants causative of BPD, as part of the NIHR BioResource-Rare Diseases Study (NIHR BR-RD). 1,213 BPD cases of uncertain genetic aetiology were recruited from 31 international centres. 687 samples were sent for whole exome sequencing and 1118 for whole genome sequencing. Cases were systematically phenotyped and genotype-phenotype relationships were assessed to detect causal variants in known and novel candidate BPD genes, including KDSR and ABCC4. KDSR is an early enzyme in the de novo sphingolipid synthetic pathway, and several cases have recently been reported with KDSR variants, severe skin pathology and thrombocytopenia, without evaluation of the mechanism of thrombocytopenia [3, 4]. I report a pedigree in which novel compound heterozygous variants in KDSR cosegregate with a severe phenotype of neonatal-onset thrombocytopenia in two siblings and juvenile myelofibrosis in the older sibling, without significant skin pathology. Cellular studies support roles for KDSR in the regulation of growth and apoptosis, megakaryocyte yield, maturity, size and proplatelet formation. This thesis also reports on the study of a pedigree in which coinheritance of homozygous loss of function variants in novel candidate BPD gene ABCC4 and heterozygous variant in known BPD gene P2RY12 cosegregate with a lifelong mild-to-moderate bleeding disorder. ABCC4 is a membrane-bound transport glycoprotein with broad substrate specificity that includes cAMP, a powerful inhibitor of platelet activation, and intra-platelet cAMP was elevated in affected individuals. The role of ABCC4 in megakaryopoiesis is also explored using CRISPR-Cas9-mediated inhibition of ABCC4 expression in cellular models of megakaryopoiesis. This study describes collaborative approaches to the analysis of high throughput sequencing data for the discovery of potentially pathogenic variants, and emphasises the necessity of functional validation of hypothetical associations.

No description available.

The complement system has a vital role both in innate and adaptive immunity. Study of diseases associated with defective complement regulation has shown the harmful effects of inappropriate and/or excessive complement activation to the host organism. The hypothesis for this thesis is that complement activation is a feature of the acute phase of several disorders, and that complement overactivation - characterized by high levels of the anaphylatoxins C3a and C5a - has a pathological role, with high levels being associated with a worse prognosis. Complement anaphylatoxins were measured in patients hospitalized with community acquired pneumonia, and patients with exacerbations of chronic obstructive pulmonary disease (COPD). Patients with thrombotic thrombocytopenic purpura (TTP) were also studied in detail: following a retrospective review of the role of immunosuppression with rituximab in these patients, a prospective study was performed to compare complement levels in acute disease with remission cases. Complement activation was present in the acute phase of all diseases studied, but there was a considerable variation in levels between cases. High systemic levels of complement anaphylatoxins were seen in several patients, but there was no clear association with prognosis; however in patients with COPD changes in sputum complement levels were associated with a longer time to clinical recovery. Patients with TTP generally had higher levels during the acute phase of the disease, and a positive correlation was found between complement activation and the level of antibody mediating the disease.

Lentiviral vectors (LVs) are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the LV components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Generation of stable packaging cell lines (PCLs) that continuously produce LVs can potentially overcome these limitations. The WinPac-RDpro cell line was developed between Collins and Takeuchi laboratories in Division of Infection and Immunity, UCL by inserting a codon-optimized HIV-1 Gag-Pol expression cassette in a continuously expressed locus in 293FT cells using Cre recombinase-mediated cassette exchange (RMCE). Subsequently HIV-1 Rev and RDpro envelope expression cassettes were serially transfected. In this thesis, WinPac-RDpro cells were used to generate model producer cells by stably transfecting a plasmid expressing a SIN GFP-encoding LV. Vector titers in excess of 106 293T transducing units (TU)/ml could be repeatedly harvested from the final producer clones in a volume of >0.5 L even under reduced serum conditions. Titers could be increased to around 1 x 10^8 293T TU/ml by concentration using scalable tangential flow filtration (TFF). Additionally, these LVs efficiently transduced human T cells and CD34+ cells at low multiplicities of infection (MOI). Titers in excess of 10^6 TU/ml were achieved using an RMCE-based strategy that was aimed at introducing a SIN LV expression cassette at a pre-selected locus. Similar titers were also achieved by using a promoterless selectable marker cloned in cis to the vector genome expression cassette. Furthermore, the Cocal Virus G protein (COCV-G) was stably expressed in WinPac cells to generate WinPac-CVG cells. These packaging cells were able to support the production of COCV-G pseudotyped SIN LVs at high titers (up to 106 TU/ml) following transient supplementation of a SIN LV expression plasmid. The efficient and stable expression of SIN LV genomes in these cells is expected to facilitate high-titer production of vectors with favorable characteristics. In conclusion, the work presented here provides significant improvements to available LV production methods. This will be of use to all basic and clinical investigators who wish to produce large batches of LVs, and addresses an important issue that has hindered large-scale LV clinical testing and application.

Chimeric Antigen Receptor (CAR) T cell therapy has had unprecedented success in the treatment of haematological malignancies, this is especially true in clinical trials targeted against CD19 for the treatment of paediatric Acute Lymphoblastic Leukaemia (ALL) and Chronic Lymphocytic Leukaemia (CLL). However, clinical evidence of on-target off-tumour toxicity was observed in anti-CD19 CAR trials as well as other CAR trials. Here I have developed a CARs platform that can potentially discriminate a cancerous tissue from normal tissue by recognizing multiple antigens presented on each cell type. Using Boolean logic we have programmed the T cell to only activate when two antigens are present (Boolean’s AND gate); only in the absence of one antigen (Boolean’s ANDNOT gate); or when both or either antigens is present (Boolean’s OR gate). We engineered the mechanism through co-localisation or kinetic segregation of the dual CARs inspired by how TCR and co-receptors work. We further found that CAR triggering responses are governed by a local balance in kinase/phosphatase activity. This platform increases the specificity and safety of the engineered T cells through the recognition of a profile of antigens rather than relying on the restricted expression of one tumour associated antigen (TAA). Logic gated CARs could reduce on-target off-tumour toxicity and open the therapy to numerous cancerous tissues where no appropriate TAAs have yet been identified.

It is not known why only certain tissues are prone to graft-versus-host disease (GVHD) injury following allogeneic hematopoietic stem cell transplantation despite widespread antigen expression. Although it is known that T cell effector pathways can have distinct effects upon individual GVHD organs, there has been no unbiased or systems-wide approach to defining the mechanisms underlying tissue-specific pathology. This thesis reports the results from a systems immunology approach to address the hypothesis that GVHD target tissues exert dominant, idiosyncratic roles in regulating effector T cell functions. To test this concept, gene expression profiles of effector CD8+ T cells infiltrating lymphoid and GVHD target organs were compared in two clinically relevant murine GVHD models. Using Weighted Gene Network Correlation Analysis, a dichotomy between the transcriptomes of T cells in peripheral tissues and lymphoid organs was identified. These profiles diverged sharply between the different GVHD target organs, and between individual sub-compartments of single organs, independently of the TCR repertoire and antigen distribution. In the skin, expression of a broad effector program was determined by the transition of T cells from the dermis to the epidermis, in a process regulated by Langerhans cells (LC). In the absence of LC, T cells were rendered incapable of up-regulating the full panoply of effector genes, showed impaired differentiation into resident memory cells and failed to induce cutaneous GVHD. By performing localized LC depletion, it was demonstrated that LC regulated T cell effector programs in situ within the epidermis by providing signals to enhance local cytokine production, promote resistance to apoptosis and enhance local survival. Collectively, these data demonstrate that GVHD is defined by tissue-autonomous regulation of effector T cells; in the skin, this is dictated by interaction with epidermal LC in situ. This work provides a rationale for precision therapies directed at blocking GVHD in individual tissues.

Endometrial cancer (EC) is the 4th commonest cancer in women in the UK. Currently histological sub-type and International Federation of Gynaecology and Obstetrics (FIGO) stage are the main determinants used in routine practice to assess a patient’s risk of recurrence after surgery, and the need for adjuvant therapies. There are currently no validated biomarkers in EC and there is an unmet need for molecular biomarkers that can accurately identify patients that are at greater risk of recurrence, and to guide treatment at relapse. In this thesis a novel biomarker panel using p53, PTEN, phospho-p70S6K, phosphostathmin, BRCA1, BRCA2, PARP1 and Rad51, has been evaluated to address this unmet clinical need. Immunohistochemistry on archived EC resection tissue has been performed using this biomarker panel and expression levels of these have been correlated to the patient’s clinico-pathological features and survival. Results demonstrate that the overexpression of p53 and phospho-stathmin and retained PTEN expression are independent predictors of overall survival (OS) and disease specific survival (DSS) in EC. Additionally, a novel liquid biopsy protocol for the identification and enumeration of circulating tumour cells (CTCs) in patients with advanced stage EC has been developed using the Parsortix device. The efficacy of this device has been compared to the Food and Drug Administration (FDA) approved CellSearch platform for enumeration of CTCs. The Parsortix device has been shown to be more efficient at enumerating CTCs and good inter-observer variability between users has been demonstrated. Further, a clinically significant cut-off level of ≥ 5 CTCs has been defined and correlated to both clinicopathological features and survival using the CellSearch platform. The expression of stathmin in CTCs (using CellSearch) has been compared to the expression of both stathmin and phospho-stathmin, in the primary tumour or biopsies taken during tumour recurrence and results have been correlated to clinico-pathological features.

Adoptive Cell Therapy (ACT) is a novel approach to cancer treatment, which implements the use of the patient T-cells redirected to eradicate tumour cells. The T-cells are engineered to express a Chimeric Antigen Receptor (CAR) that bestows specificity for an arbitrary antigen. CAR-T cell clinical trials have shown tumour eradication of leukaemia, but modest results against solid tumours. There is an ongoing debate regarding the correlation of CAR affinity and antigen-density recognition threshold, which we sought to investigate. Although the TCRs sensitivity against low antigen targets is superior at low-affinity, the effect of affinity in CAR efficacy and sensitivity is controversial. In order to examine the correlation between CAR efficacy and affinity, we designed a CAR against an intracellular protein in melanoma. Tyrosinase-Related Protein 1 (Tyrp1) resides in the surface of the organelles called melanosomes. However, Tyrp1 is also trafficked to the surface at a low density. We mutated an anti-Tyrp1 single-chain Variable Fragment (scFv) to acquire eight mutants of various affinities. The affinity range was 0.74nM-54.3nM. Including the wild-type scFv, these nine affinity gradient CARs were challenged with a cell line expressing low, medium and high densities of cell-surface Tyrp1. Three mutant scFv CARs were superior to the wild-type scFV. However, the affinities of those superior CARs was variant. There was no pattern observed to suggest a direct correlation between the affinity and the CAR efficacy. In contrast to previous publications, this study shows that affinity does not play a key role in determining the CAR efficacy. We hypothesise that the difference in CAR efficacy depends on a combination of factors, such as scFv stability and affinity, CAR density, and antigen density. However, the complex interaction of these parameters, as well as further confounding factors, renders the deduction of a pattern challenging. In order overcome this complexity, a multivariate analysis of all the parameters together is necessary.

The linear ubiquitin chain assembly complex (LUBAC) modulates signalling outcomes of the tumour necrosis factor receptor 1 signalling complex (TNFR1-SC) by placing linear ubiquitin linkages on target proteins in this complex, which in turn leads to recruitment of effector and regulatory molecules. Thereby, LUBAC promotes TNF-induced gene-activatory signalling. Furthermore, LUBAC activity is crucial for the protection from TNF-induced cell death by limiting the formation of the death-inducing cytoplasmic signalling complex II. This thesis demonstrates that HOIP, the enzymatically active subunit of LUBAC, in its function as an adaptor or via its enzymatic activity, orchestrates the recruitment of a multitude of TNFR1-SC components and thereby specifically regulates TNFR1-SC signalling outcome with respect to gene-activatory signalling and cell death. The deubiquitinases CYLD and A20, respectively recruited by LUBAC or linear ubiquitin chains, cooperatively restrict gene activation but exert opposing effects on M1-linked ubiquitin chain stability and, thereby also on cell death. Whilst CYLD-mediated cleavage of M1 chains sensitizes cells to TNF-induced cell death, A20 binding to them prevents their hydrolysis which inhibits cell death. Furthermore, it is shown that the IKK-related kinases TBK1 and IKKε are recruited to the TNFR1-SC in a LUBAC activity-dependent manner. Whilst TBK1 can be recruited via the adaptors TANK or NAP1, IKKε recruitment to the TNFR1-SC solely requires TANK. Importantly, inhibition of the kinase activity of TBK1 and IKKε or abrogation of their recruitment to the TNFR1-SC results in sensitization to TNF-induced RIP1-activity-mediated cell death. Mechanistically, these non-canonical kinases prevent RIP1 from transitioning to complex II and, thereby, from triggering apoptosis or necroptosis. In summary, this PhD thesis elucidates how LUBAC regulates TNF-induced signalling outcomes in terms of gene-activatory signalling and cell death. In doing so, it identifies a previously unrecognised molecular mechanism which facilitates the recruitment and activation of the non-canonical kinases TBK1 and IKKε at the TNFR1-SC.